AU663519B2 - Method for the fermentative production of cephalosporin C using acremonium chrysogenum - Google Patents

Abstract

Described is a method for the fermentative production of cephalosporin C, the fermentation solution being filtered during fermentation through a cross-flow filtration system. The filtrate obtained from the fermenter can be used again.

Description

OPI DATE 16/03/93 APPLN. ID 24024/92 AOJP DATE 27/05/93 PCT NUMBER PCT/EP92/01811 II1I1111111111111111 I~II~ ~IN AU9224024 INTERN ATION ALE ZUSAMMENARBEIT A UF DLM GEBIE L)LDS P)A I EN' I %t SENS (Ilj I) (51) Internationale Patent klassi fi kation 5 (It) Internationale X'er~ffentlichiingsnumnier: WO 93/04188 C12P~ 3506A (43) Internationales C1P3/6Vcrtffentlichungsdatuni: 4. Mfirz 1993 (04.03.93) (21) Internationales Aktenzeichien; PCT,'EP92/0181 1 (74) Genleinsaner Vertreter: HOECHST AKTIENGESELL- SCHAFT: Zentrale Patentabteilung, Postfach 80 03 (22) Internationales Anmeldedatumn: S. August 1992 (08.08.92) D-61-30 Frankfurt am Main 80 (DE).

Priorit~tsdaten: (81) Bestimmunusstaaten: AU, CA. CS. Fl, HU, JP, KR, NO, P41 27 648.5 21. August 1991 (21.08.91) DE RU, US, europaisches Patent (AT, BE, CH, DE, DK.

E S. FR, GB, GR, I E, IT, LU, MC, NL, SLE).

(71) Anmelder (fi, alle BL'sitnnulrgsstaaien ausser USi: HO- ECHST AKTI ENGESELLSCHAFT [DE 'DEl; Post- Veriiffentlicht fach 80 03 20, D-6230 Frankfurt am Main 8(0 Alit iflterfatwflalem ReclzLrchenberilclu (72) Erfinder; und Erfinder/Anmelder (nur Iiir LIS) BAYER. Thomas IDE'" DE1 Bismarckstrage 14, D-6232 Bad Soden am Taunus (E.SCHRAMM, Wilhelm Arnselweg 18.

D-6233 Kelkheim RATHSCHECK, Wolfgang~ Carl-Orff-Weg 13. D-6232 Bad Soden am Taunus (DE).

(54) Title: METHOD FOR THE FERMENTATIVE PRODUCTION OF CEPHALOSPORIN C USING ACREMONIUM

CHRYSOGENUM

(54) Bezeichnung: VERFARREN ZUR FERMENTATIVEN HERSTELLUNG VON CEPHALOSPORIN C MIT ACREMO- NIUM CHRYSOGENUM (57) Abstract Described is a method for the fermentative production of cephalosporin C, the fermentation solution being filtered during fermentation through a cross-flow filtration system. Thle filtrate obtained from the fermenter can be used again.

(57) Zusamnmenfassung Es wird emn Verfahren zur fermentativen Herstellung von Cephalosporin C beschrieben, wvobei die Fermentationsl~sung wiihrend der Fermentation fiber emn Cross- Flow- Filtrationssystem filtriert wird. Die abgenommene Filtratmenge aus dem Fermenter kann wieder eingesetzt werden.

HOE 91/F 262-Dr.TH/sch 1 Description Process for the fermentative production of cephalosporin C using Acremonium chrysogenum Antibiotics-containing culture liquids are generally worked up after the maximum content has been reached. In most cases, the first step is a separation of cells and solids by filtration or by centrifugation. This may be followed by concentrating the valuable constituents by extraction or by adsorption. As a rule, the purified product is then crystallized and can be processed to give semisynthetic antibiotics.

A disadvantage in this step-by-step method is the instability of a large number of antibiotic molecules such as, for example, cephalosporin C (CPC). CPC is already degraded during the fermentation. This can be effected either purely chemically, by water attacking the g-lactam ring, or enzymatically, for example by esterases (Konecny et al., 1973, J. of Antib., 26, 3, 135-141). A series of secondary products such as deacetoxycephalosporin C (DOCPC) and deacetylcephalosporin C (DCPC), which must be removed from the CPC during the purification step, is also formed during the fermentation. This results in considerable yield losses. Cross-flow filtration systems using polymer or ceramics membranes have already been used for the working-up of culture liquids which contain antibiotics (Harris et al., J. Chem. Techn. Biotechnol., 1988, 42, 19-30). A reduced decomposition of Scephalosporin C in these processes has not been demonstrated as yet. It is known to isolate cephalosporin C after the fermentation using cross-flow filtration (M.

Kalyanpur et al., Contributed Paper, Chapter 32, pages 455-470).

Ti It is an object of the present invention to find a S process in which the degradation of cephalosporin C and REPLACEMENT SHEET HOE 91/F 262-Dr.TH/sch 2 the formation of secondary products can be reduced and 4 1k' REPLACEMENT SHEET

I*~

'-7 2 e0L_ the yield relative to the amount of substrate employed can be increased.

Surprisingly, it has now been found that the use of a cross-flow filtration module during the fermentation of Acremonium chrysogenum allowed the CPC yield to be increased, the formation of DCPC to be reduced and the production time to be extended. The filtration and the fact that small amounts of DCPC are formed furthermore simplify the working-up of cephalosporin C substantially.

The invention therefore relates to a process for the preparation of CPC which comprises filtering the fermentation solution during the fermentation through a crossflow filtration system and the possibility of replacing the withdrawn amount of filtrate in the fermenter.

Acremonium chrysogenum (Cephalosporium acremonium) as well as mutants and selectants, as long as they produce CPC derivatives, are suitable for use in the process according to the invention.

The nutrient solution contains carbon sources such as sucrose, corn starch, dextrose or molasses, and nitrogen sources such as soybean meal, groundnut meal, malt extract or ammonium acetate.

The nutrient medium also contains inorganic salts such as sodium hydrogen phosphate, sodium chloride, calcium chloride, calcium sulfate, calcium carbonate, magnesium sulfate or potassium hydrogen phosphate. Fat such as Smethyl oleate or soybean oil can also be added to the nutrient medium. Trace elements such as salts of iron, manganese, copper, zinc, cobalt or other metals are also added.

Acremonium chrysogenum (Cephalosporium acremonium), preferably DSM 6473, is cultured at temperatures between 0 C and 30 0 C, preferably at 25 0 C, and at a pH of between S and 8, preferably pH 7. It is first cultured REPLACEMENT SHEET j

F.

S- 3aerobically in a shaker flask and then in a fermenter with stirring and aeration with air or pure oxygen. The microorganisms are cultured' in the fermenters over a period of from 120 to 240 hours, preferably between 130 and 170 hours.

The cross-flow filtration systems used can be plate modules, tube modules, capillary modules, wound modules or hollow-fiber membrane modules made of polymers, carbon or cera-i.cs and having separation borders from the ultrafiltration to the sterile-filtration range. Filtration modules which are preferably employed are those which have a pore size of 0.2 gzm moX4 nm. The membrane materials which can be employed are polysulfones, polyamides, cellulose acetate, aluminum oxide or zirconium oxide. The filtration can be affected continuously or batchwise. It starts approximately 2 3 days after inoculation of the fermenter and can be continued until the fermentation has ended. The flow rate of the fermenter solution over the filtration surface is between and 20 m/s, preferably 1 to 10 m/s.

The filtrate which is drawn off from the fermenter during the fermentation can be replaced by a corresponding amount of liquid or, alternatively, the filtrate can be recycled to the fermenter after the valuable product has been separated for example by absorption. To this end, water, enriched with suitable salts or other nutrient medium components, can be pumped in.

The liquid volume which has not permeated the membrane is recycled to the fermenter.

The fermenter and the cross-flow filtration system are connected to suitable pipes or tubes and sterilized before the fermentation. Approximately 0.2 m 2 of filtration surface are required for a 100 1 fermenter. It is also possible to use greater and smaller filtration surfaces.

REPLACEMENT SHEET -i 4 Example 1: i' Fermentation of Acremonium chrysogenum DSM 6473 The fermentation was carried out using the following nutrient solution: Preculture medium g/1 Corrsteep 11.75 Ammonium acetate Sucrose 20.0 CaSO 4 2H 2 0 MgSO 4 7.HO pH 7.0 (adjusted using 15 by wt. NaOH) Fermentation medium g/1 Fat-free groundnut meal 100.0 Ammonium acetate Glucose monohydrate Methyl oleate D,L-Methionine CaSO 4 2HO0 MgSO 4 7.H 2 0 CaCO, Antifoam Fed-batch solution: Glucose monohydrate 500.0 D,L-Methionine 24.75 Agar slants are used for inoculating 100 ml of preculture medium (500 ml shaker flasks equipped with 4 baffles).

The flasks are incubated for 48 hours at 150 rotations per minute (rpm) at 25 to 28 0 C. These cultures are used for inoculating a further preculture (1000 ml of medium, 5000 ml flasks, 25 to 28 0 C, 120 rpm, 58 to 60 hours). The second preculture is used for inoculating 60 1 REPLACEMENT SHEET I I 5 fermentation medium in a stirred fermenter. The fermentation is carried out at 25 0 C. The gas flow is controlled in such a way that the pO 2 in the fermentation nutrient solution is above 20 After 74 hours fermentation, the filtration is started using a cross-flow ceramics module (manufactured by Membraflow, a-Al,0o) having a filtering surface of 0.2 m 2 and a pore size of 0.2 Am. The following process parameters were adhered to: Flow rate: Pumping rate: Filtration capacity: Filtration time: 2 m/s 1500 1/h 2 1/h 68 hours.

The table which follows shows the test results after 142 hours of a fermentation without and with (B) cross-flow filtration: Table 1: CPC DCPC/ Productivity CPC Productivity CPC

DCPC

max. after 6 after 6 days days A 100 100 100 100 100 B 140 44 200 400 53 Example 2 The fermentation is as in Example 1. Table 2 shows the results after 167 hours of a fermentation with cross-flow filtration compared with a parallel fermentation without filtration, which was stopped after 142 hours: REPLACEMENT SHEET II -6 Table 2: CPC DCPC/CPC M% M% (142 hours) 100 100 B (167 hours) 129 69 REPLACEMENT SHEET

Claims (8)

1. A process for the fermentative production of cephalosporin C using Acremonium chrysogenum, which comprises filtering the fermentation solution including Acremonium chrysogenum during the fermentation through a cross- flow filtration system to obtain cephalosporin C and the possibility of replacing the amount of filtrate in the fermenter.

2. The process as claimed in claim 1, wherrin a polymer filter or ceramic filter is used as the cross-flow filtration system.

3. The process as claimed in claim 1 or 2, wherein the filter has a pore size of between 4 and 200 nm.

4. The process as claimed in one or more of claims 1 to 3, wherein the flow rate over the filter surface is 1 to 10 m/s. The process as claimed in one or more of claims 1 to 4, wherein the filtrate solution in the fermenter can be replaced by water.

6. The process as claimed in claim 5, wherein the filtrate solution is replaced by a nutrient solution.

7. The process as claimed in claim 6, wherein the filtrate solution is recycled to the fermenter after the valuable product has been removed. t L l 1 z Ih,-

8- Abstract of the disclosure HOE 91/F 262 Process for the fermentative production of cephalo- sporin C using Acremonium chrysogenum There is described a process for the fermentative produc- tion of cephalosporin C in which the fermentation solu- tion is filtered during che fermentation through a cross- flow filtration system. The amount of filtrate which has been drawn off from the fermenter can be replaced. INTERNATIONAL SEARCH REPORT International application No. PCT/EP 92/01811 A. CLASSIFICATION OF SUBJECT MATTER Int.C1. 5 C12P35/06 According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) Int.C1. 5 C12P Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X Contributed Paper, chapter 32, pages 455 470, 1-8 M. Kalyanpur et al: "Isolation of Cephalosporin C from Fermentation Broths Using Membrane Systems and Hig-Performance Liquid Chromatography", see page 456, lines 16-26 X J. Chem. Tech. Biotechnol., Vol. 42, 1988 Tim A.J. 1-8 Harris et al.: "The Cross-flow Filtration of an Unstable Beta-lactam Antibiotic Fermentation Broth", see page 19 page see in particular page 20, lines 13-26 O Further documents are listed in the continuation of Box C. See patent family annex, Special categories of cited documents: later document published after the international filing dateor priority document defining the general stateof the art which is not considered date and not in conflict with the application but cited to understand to be of particular relevance the principle or theory underlying the invention earlier document but prlished on or after the international filing date document of particular relevance; the claimed invention cannot be considered novel or cannot be considered to involve an inventive document which may ,trow doubts on priority claim(s) or which is step when the document is taken alonean iveve cited to establish the publication date of another citation or other step when the document is taken alone special reason (as specified) document of particular relevance; the claimed invention cannot be document referring tc an oral disclosure, use, exhibition or other considered to involve an inventive step when the document is means combined with oneor more othersuch documents, such combination "P document published prior to the international filing date but later than being obvious to a person skilled in the art the priority date claimed document member of the same patent family Date of the actual completion of the international search Date of mailing of the international search report 26 October 1992 (26.10.92) 11 November 1992 (11.11.92) Name and mailing address of the ISA/ EUROPEAN PATENT OFFICE Facsimile No. Authorized officer Telephone No. Form PCT/ISA/210 (second sheet) (July 1992) INTERNAnrONALrR I sERCHENBERICHIT Internationates Aktenzelchen PCT./EP 92/01811 1. KLASSIFIKATI0N DES ANMELDUNGSGENSTANDS (bei menraren Klassilkaticnssymbolen sind alto ;nzugeben)Q Nach der Internationalen Patentktassitikation (IPC) Oder nach der nationalen Klasssitlkation und der IPC itnt.CI. 5 C 12 P 35/06 If. RECI4ERCHIERTE SACHGEBIETE Recherchierter M indestprils tall 7 Klassitikationssystem IKlassifikationssymbole Inrt.CI G 12 P Recherchierte nicht zumn Mindestpriifslotl gehbrande Verbttentlictungen, soweit diese unter die recherchlerlen Sacligebiete fallent Ill. EINSCHLAGIGE VEROFFENTLICHUNGEN 9 Art Keneziclinung der Verblienttichung ,soweit ot-torderlich unter Angabe der maebtichen Teite 2 er f~fJhN. X Contributed Paper, Kapitel 32, Seiten 455-470, 1-8 M. Kalyanpur et al: "Isolation of Cephalosporin C from Fermentation Broths Using Membrane Systems and High-Performance Liquid Chromatography", Siehe Seite 456, Zeilen 16-26 X J. Chem. Tech. Biotechnol., Band. 42, 1988 Tim A.J. 1-8 Harris et al.: "The Cross-flow Filtration of an Unstable Beta-lactam Antibiotic Fermentation Broth", siehe Seite 19 Seite Siehe insbesondere Seite 20, Zeilen 13-26 Besondore Kategorien von angegebenen Verb I entl ich ungen ''S~eeVrletihndenc e nento~e n Verditentlichung, die den atlgomoeinen Stand der Technik TS~eeVrtetihndenc e nontoae n detiniert, abor nicht ats besonders bedeutsam anzusehen ist meldedntum Odor dem Priorititsdatum verbttentlicht worden jot und mit der Anmeidung nicht kotlidiert, sondern nur zumn E12 5tores Dokument, das jedoch orsl am Oder nach dem interna- Vorstindnis des der Erfindung zugrundetiegenden Prinzips tionaten Anmetdedatum verbitentticht worden ist Oder der ihr zugrundeliegenden Theonie angegeben ist Verbttenttichung, die geeignet ist, einen Priori t5Isans pruch IX' Vertientlichung van besondorer Bedeutung, die beanspruch- zwoitethatt erscheinen zu tassen, Oder durch die dos VeriiI- te Erfindung kann nicht als neu Oder aut ertindenscher Titig- tenttichungsctatum elner anderefl im Rocherchenborlcht ge- koit boruhend betr-achtet werden nannten Verii~tentlichung bolegt werden soil Oder die aus ein- em andoron besonderen Griond angegeben Ist (Wie ausgettthrt) Y' Verotfentlichung von besonderer Bodeutung, die beanspruch- te Ertindung kann nicht als auf erlinderischer Tfitigkceit be. Verbltentlichung, die sich aul eine mgndliche Oftenbarung, ruhond betrachtet werdon, wenn die Verbffentlichung mit eine lGenutzung, eine Aussteltung Oder andere Mainahmen einer Oder mehreren anderen Verbifentlichungen dieser Kate- bezleht gonie in Verbindung gebracht wird und diese Verbindung f~r 'P Verditontlichung, die vor demn internatlonalen Anmeldeda- omnen Fachmann nahetiegend lat tumn, abor nach dem beanapruchton Priorititsdatum verbiment- VorbIfeetlchung, die Mltgtied deirselbon Patentiamitie ist licht worden ist IV. BESCHEINIGuNG Datum des Abschtusses der internationaten Recherche Absondedatumn des internationalen Rocherchenbeichts

26. "Dktober 1992 :1 1 NO0V1 199 tn',emnationate Rocnerchenbehbrdo Unterschrilt des bevolmichtigton Bodienstoton EurapAiscttes Patentami na jln Formbtatt PCT/ISA/210 (Btatt 2) (Januar 1 985) I